68.2
Journal of Natural Products V O ~4.7 , N O .4 , pp. 682-686, J u l - A ~ g1984
DOPAMINE BIOSYNTHESIS AT DIFFERENT STAGES OF PLANT DEVELOPMENT IN P A P A V E R S O M N I F E R U M KATHRYNK. KAMO' and PAULG. MAHLBERG Department of Biology, Indiana University. Bloomington, IN 47405 ABsTRAcT.-Latex and cell-free extracts of various organs and stages of plant and capsule development in Papaver somn+rum, the opium poppy, synthesized dopamine, an alkaloid precursor, from '*C-dopa. The 1000 g X 30 min supernatant from latex of the pedicel-capsule junction converted more dopa than latex supernatant from the upper capsule or lower pedicel regions, although there was more protein in the latex from the capsule. Percent conversion of pedicel-capsule latex into dopamine was maximum in unopened flower buds and decreased within 14 days after flowering. Dopamine biosynthesis in latex and cell-free extracts also varied with the stage of organ development. Extracts from capsule tissue converted more labeled dopa into dopamine than did extracts from pedicels, leaves from vegetative plants at the rosette stage, leaves from flowering plants, or pedicels connected to capsules.
L-3-4 Dihydroxyphenylalanine (dopa) and dopamine have been established as alkaloid precursors in intact plants of Papaversomni$erzm L. (1-3). When 2-14C-dopa or 114C-dopamine was injected into intact poppy capsules, all of the radioactive label, upon extraction and degradation of the labeled morphine, was specifically at the ethanamine bridge position. Laticifer cell protoplasm has been studied as a site of alkaloid biosynthesis, with 14 C-dopa as the alkaloid precursor. There was evidence for the presence of dopa decarboxylase in the 1000 g X 30 min latex supernatant fraction that converted dopa to dopamine (4,5). The 1000 g X 30 min latex pellet fraction synthesized five major alkaloids-morphine, codeine, thebaine, papaverine, and narcotine-when the pellet was incubated wirh labeled dopa (6,7). In addition, Papaver alkaloid biosynthesis has been studied in cell-free systems (811). There are several advantages of a cell-free system for studying biosynthetic reactions. With such an extract, one avoids the problems of precursor penetration into the plant tissue, translocation within the plant, possible subcellular compartmentation of metabolic pools (12), and as in this study, cell-free extracts allow one to study laticifer protoplasm in conjunction with other tissues from regions of the plant, such as the leaves, where it is difficult to collect substantial amounts of latex for in vitro latex studies. The alkaloid and dopamine concentration in poppy organs varies during stages of plant development and in different organs ( 7 , 1 5 2 1). Recently, it was reported that latex collected from plants in the floral bud stage contained higher levels of dopamine than did latex collected 3-12 days after the plants had flowered. These variations in alkaloid concentration may reflect translocation of an alkaloid or its precursor to a particular plant organ rather than differences in alkaloid biosynthetic activity for various organs at different stage of plant development. The purpose of this study was to examine the capacities of isolated latex and cell-free extracts to synthesize dopamine, an alkaloid precursor, from 14C-dopa. Various organs and tissues at different stages in development were examined to evaluate dopamine synthesize in both the latex and cell-free systems. 'Present address: Botany and Plant Pathology, Lilly Hall, Purdue University, West Lafayette, IN 47907.
Jul-Aug 1984} Kamo & Mahlberg:
Dopamine Synthesis in Papaver
683
EXPERIMENTAL ANALYSIS OF LATEX FOR DOPAMINE BIosYNTHEs1s.-The pedicel-capsule was lanced when submerged in buffer (0.5 M mannitol, 0 . 1 M phosphate, p H 7.2) on ice so as to prevent latex oxidation. In all experiments, the 1000 g X 30 min latex supernatant was used because dopa decarboxylase occurs only in the supernatant (4,5). The dopamine assay method for incubating latex with "C-dopa and the preparation ofsamples for the Immediately after latex collecCG-50 column were similar to the procedure of Roberts and Antoun (4). tion, the latex supernatant was added to a tube containing 0.18 FCi [3-'*C]dopa, specific activity 1.85 mCi/mmole (Amersham), previously dried down in the tube with N,. The tubes were swirled, gassed for 3 min with N,, and then incubated at 35" for 20 min. For controls, the latex was boiled for 30 min. Samples were stored at -20" until ready to run on the ion-exchange column. The column (Pasteur pipet) was packed with 6 cm ofprepared (22,23) Amberlite CG-50 resin. Samples (3 ml each) were loaded onto the column and chromatographed with 15 ml of 0.05 M ammonium acetate, p H 6.2 followed by 20 ml of 1 M ammonium acetate, p H 6.2. The dopamine fraction (the first 12 ml) of the 1 M ammonium acetate pH 6 . 2 wash were collected for analysis. The percent conversion of '*C-dopa to dopamine was calculated using liquid scintillation. Dpm was calculated by internal standardization using D-[U-"Clglucose. specific activity 4.5 mCi/mmole. The dopamine concentration was determined from an aliquot of the 1 M ammonium acetate dopamine-containing fraction. Total dpm was calculated from all fractions obtained from the ion-exchange column. The dopamine product collected from the ion-exchange column was verified by two-dimensional pc on Whatman No. 1 chromatography paper. The solvent system was n-PrOH-H,O-n-BuOH (5:7:10) (4) for the first dimension and H,O-n-BuOH-pyridine (1: 1: 1)(24) for the second. Dopamine was identified by its Rf values in the two different solvent systems and its color when sprayed with a mixture of equal volumes of 15% ( w h ) FeCI, and 1% ( w h ) K,Fe(CN), (25). Autoradiograms were prepared from the paper chromatograms to determine the presence of labeled dopamine and the possible presence of contaminants. Paper chromatograms were sprayed with Enhance Surface Spray (New England Nuclear) and then exposed to K0dak.X Omat AR, XAR-5 film at -70". PROTEINDETERMINATION.-The protein in the latex sample was determined according to the method of Lowry (26) using latex supernatant diluted with buffer. ANALYSIS O F CELL-FREE SYSTEM FOR DOPAMINE BIOSM'JTHESIS.-This assay was based upon the methods of Roberts and Antoun (4) and Hodges and Rapoport (11). Plant organs were homogenized in a phosphate buffer ( 0 . 1 M phosphate, 10 mM DIECA, 20 mM ascorbic acid, p H 7.2) to prepare the cell-free extract. Specific organs (capsules, pedicels, and leaves from plants at the rosette or flowering stages) were obtained from plants up to 5 days after flowering, except for leaves from vegetative plants in the rosette stage of growth. Flowering was defined as the first day the corolla opened. The cell-free filtrate was prepared from homogenized tissue filtered through #80 mesh nylon cloth (Tetko Company). The 3-ml incubation mixture included 2.5 ml of the filtrate and 0.51 FCi L-13'*C)dopa, specific activity 1.7 mCiimmole. The reaction was initiated by adding "C-dopa (1 X lo-* M) diluted with 0 . 1 M phosphate buffer, p H 7.2 buffer. The tubes were incubated at 35" for 60 min, and the tubes were inverted every 20 min during the incubation period. Tissue boiled for 30 min served as the control. The reaction was terminated by adding 3% ( w h ) sulfosalicylic acid (0.2 ml), 5% (w/v) EDTA (0.2 ml), and 5% (wiv) sodium metabisulfite (0.2 ml). Cold dopamine carrier (2 mg) was added to the incubation mixture. Protein was precipitated by 10% TCA followed by centrifugation at 4500 g X 15 min. The p H of the supernatant was titrated to p H 6.2 with ",OH. Each sample volume was brought to 6 ml with the 0 . 1 M phosphate buffer. Samples were kept at -20" until they were run on the ion-exchange column as for latex samples. The calculation of "C-dopa percent conversion to dopamine was determined by liquid scintillation using the method as for latex samples. In addition, the method for analyzing dopamine by two-dimensional chromatography and autoradiography was identical to that used for examining the dopamine samples from latex. PLANTCULTIVATION.--P. Iornniferum plants were grown from seeds (UNL 376 from Rennes, France) in a greenhouse or outside when weather permitted. Plants were grown 2 months under short-day conditions (8 h light and 16 h dark) and subsequently induced to flower under long-day conditions (15 h light and 9 h dark) using incandescent light.
RESULTS AND DISCUSSION REthis comparative study of dopamine biosynthesis, latex was collected from
DOPAMINE BIOSYNTHESIS IN LATEX COLLECTED FROM DIFFERENT PLANT
GIONS.-In
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three regions of the floral axis: the capsule, the pedicel-capsule junction, and the pedicel base region. Equal amounts of latex were collected from plants within 5 days after flowering. DIECA was added to the supernatant to inhibit dopa oxidation by polyphenol oxidase. The supernatant may have been contaminated by this enzyme, which is localized in the 1000 g X 30 min pellet (5). Our results agreed with Roberts (5) that polyphenol oxidase was present in the 1000 g X 30 min pellet, while dopamine biosynthetic activity was in the 1000 g X 30 min latex supernatant fraction. Incorporation experiments showed more conversion of 14C-dopa into dopamine in latex ofpedicel-capsule origin than in latex from other regions (Table 1). Latex from the pedicel-capsule region converted nearly twice as much dopa as did the latex from the lower pedicel position. These results were unexpected because there was considerably more protein in latex from the capsule than in latex from the pedicel-capsule position (Table 1). Latex from the three different regions differed in physical appearance, protein concentration, and dopamine biosynthetic activity. TABLE1. Latex from Different Regions of the Plant Analyzed for "C-dopa Conversion into Dopamine" I
I
Plant part Capsule . . . . . . . . . . . . . . . Pedicel-capsule junction . . . . . . . Lower pedicel . . . . . . . . . . . .
4,428 6,432 3,036
I
Conversion
mg proteidml diluted latex
1.26 1.79 0.93
130 42 40
The presence of labeled dopamine in the dopamine fraction collected from the CG50 ion-exchange column was verified by two-dimensional pc. The Rf values (0.39 in the first dimension and 0.69 in the second dimension) and the purple color in response to FeC13-K3Fe(CN)6 spray were similar for both the sample and standard dopamine. The autoradiogram of the sample contained only one radioactive spot with the same Rf as standard dopamine in the two solvent systems. A 3-week exposure period was optimal for the chromatograms. Exposures for 2 and 6 weeks showed no other radioactive compounds present on the autoradiograms. The same results were obtained for the control autoradiograms, which showed no radioactive spot for dopamine. Counts in the dopamine control fraction were negligible (50-60 dpm).
DOPAMINE BIOSYNTHESIS IN LATEX COLLECTED FROM CAPSULES OF DIFFERENT AGEs.-ktex was collected from the pedicel-capsule junction of floral buds several days prior to flowering, and subsequently 1-2, 5-6, and 13-14 days after flowering. From each age the latex of 40 plants set was pooled. Protein determinations showed the latex from these developmental stages contained approximately the same amount of protein, 25-30 m g proteidml diluted latex (Table 2). The conversion of '*C-dopa ranged from 0.8 to 2.1% (Table 2). More incorporation of labeled dopa occurred in latex from capsules of the preflowering stage than in latex collected 1-14 days after flowering. Thus, dopamine biosynthetic activity in latex declined as the capsule matured. This result is in agreement with Roberts (5) who reported that latex before flowering contained higher levels of dopamine than latex after flowering. Because our results indicated that preflowering latex had higher dopamine biosynthetic activity, the dopamine levels reported by Roberts may be the result of
Jul-Aug 17841 Kamo & Mahlberg:
Developmental stage Before flowering . . . . . 1-2 days after flowering . 5-6 days after flowering . 13-14 days after flowering
Dopamine Synthesis in Papaver
Conversion
DPm
. . . . . . . . . . . . . . . . . .
. . . . . .
6,576 4,296 5,508 3,348
685
(rc)
mg proteidml diluted latex
2.1 1.3 1.7 0.8
26 30 25
27
"The incubation mixture contained 1000 g X 30 min latex supernatant, 0.18 pCi I4C-dopa (1.85 mCiimmole), 0 . 1 M phosphate, 0.5 M mannitol, 20 mM ascorbic acid, 10 mM DIECA buffer, p H 7.2 to a total volume of 0.5 ml. Incubation was for 20 min at 35". The values listed represent the average of two samples, and each sample was collected from the pedicel-capsule of 40 plants.
dopamine biosynthesis rather than increased dopamine translocation to the capsule before flowering or of a dilution of dopamine in latex after flowering when the capsules and its laticifers rapidly expand. DOPAMINEBIOSYNTHESIS IN CELL-FREE EXTRACTS DERIVED FROM VARIOUS PLANT ORGANS.---cell-free extracts were prepared from: leaves at the rosette stage of vegetative plants, leaves from flowering plants, capsules, pedicels, and capsules connected to pedicels. These extracts were incubated with '*C-dopa followed by ion-exchange and paper chromatography. An autoradiogram of the two-dimensional paper chromatogram from the cell-free sLmple showed one radioactive spot with the same Rf values as standard dopamine. This radioactive spot turned purple when sprayed with the solution of FeCI3 and K3Fe(CN)6,indicating a positive reaction for dopamine. The control, also analyzed by two-dimensional pc, contained no labeled dopamine and had negligible counts (around 60 dpm). Dopamine biosynthesis varied in cell-free extracts derived from various organs and different stages of plant growth. Extracts from leaves of the rosette stage converted more L-dopa (0.95%) than did leaves of flowering plants (0.38%)(Table 3). Maximal conversion ( 1.70%) occurred in cell-free extracts from capsules. This study has demonstrated that the plant organs examined (leaves, capsules, and pedicels) possessed dopamine biosynthetic activity. Leaves of young plants in the rosette stage converted dopa to dopamine. After the plants matured and flowered, the leaves progressively lost this ability, but the capsules began to synthesize dopamine. TABLE3.
"C-Dopa Conversion into Dopamine for Cell-Free Extracts of Different Organs" Dopamine Fraction
Origin ofcell-free Extract
Capsules . . . . . . . . . . . . . . Pedicels . . . . . . . . . . . . . . Capsules connected to pedicels . . . . Leaves rosette stage . . . . . . . . . Leaves flowering stage . . . . . . . .
i I
Conversion
Dpm 20,412 5,244 17,136 8,748 4,442
~
(%I 1.90 0.58 1.48 0.95 0.38
"The cell-free system contained cell-free extract (2.5 ml), 0.5 pCi L-dopa (1.67 mCiimmole), 10 mM DIECA, 20 mM ascorbic acid and 100 mM phosphate buffer, pH 7.2 to a total volume of 3.0 ml. Incubation was for 60 min at 35". Each value listed in the table represents the average of two samples, and each sample consisted of 10-35 plant organs depending on the plant part.
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Examination of isolated latex showed that the greatest dopamine synthesis occurred prior to flowering. The dopamine synthetic capacity of latex declined as the capsule matured to 14 days after flowering. The differences in dopamine biosynthesis for various tissues and for tissues of different ages emphasize the importance of relating biosynthetic reactions to plant development. ACKNOWLEDGMENTS The authors thank the United Nations Narcotics Laboratory for the P. somnifeum seeds. Dr. John McCain is thanked for reading the manuscript. The research was supported by grants from Sigma Xi, Indiana Academy of Science, and USDA funds (53-32R6-070). LITERATURE CITED 1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2 1. 22. 23. 24. 25. 26.
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Received 22 November I983
